Microbiology

1. PowerPoint® Lecture
Presentations prepared by
Mindy Miller-Kittrell,
North Carolina State University
C H A P T E R
© 2015 Pearson Education, Inc.
Microscopy,
Staining, and
Classification
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Microscopy and Staining: Overview

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Units of Measurement
• Tell Me Why
• Why do scientists use metric rather than English units?

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Microscopy
• Microscopy: the use of light or electrons to
magnify objects
• Science of microscopy begun by Antoni van
Leeuwenhoek
• Various types of light and electron microscopes

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Microscopy
• General Principles of Microscopy
• Wavelength of radiation
• Magnification
• Resolution
• Contrast

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Figure 4.1 The electromagnetic spectrum.

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Figure 4.2 Light refraction and image magnification by a convex glass lens.
Light
Air Glass
Focal point
Specimen Convex
lens
Inverted,
reversed, and
enlarged
image

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Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of
various types of microscopes.

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Microscopy
• General Principles of Microscopy
• Contrast
• Differences in intensity between two objects or between
an object and its background
• Important in determining resolution
• Staining increases contrast
• Use of light that is in phase increases contrast

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Microscopy
• Light Microscopy
• Bright-field microscopes
• Simple
• Contain a single magnifying lens
• Similar to magnifying glass
• Leeuwenhoek used simple microscope to observe
microorganisms

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Microscopy
• Light Microscopy
• Bright-field microscopes
• Compound
• Series of lenses for magnification
• Light passes through specimen into objective lens
• Oil immersion lens increases resolution
• Have one or two ocular lenses
• Total magnification = magnification of objective lens x
magnification of ocular lens
• Most have condenser lens (direct light through
specimen)

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Figure 4.4 A bright-field, compound light microscope.
Coarse focusing knob
Moves the stage up and
down to focus the image
Illuminator
Light source
Diaphragm
Controls the amount of
light entering the condenser
Condenser
Focuses light
through specimen
Stage
Holds the microscope
slide in position
Objective lenses
Primary lenses that
magnify the specimen
Body
Transmits the image from the
objective lens to the ocular lens
using prisms
Ocular lens
Remagnifies the image formed by
the objective lens
Line of vision
Ocular lens
Path of light
Prism
Body
Objective
lenses
Specimen
Condenser
lenses
Illuminator
Fine focusing knob
Base
Arm

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Figure 4.5 The effect of immersion oil on resolution.
Glass cover slip
Slide
Specimen Light source
Without immersion oil
Lenses
Immersion oil
Glass cover slip
Slide
Light source
With immersion oil
Microscope
objective
Refracted light
rays lost to lens
Microscope
objective
More light
enters lens

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Microscopy
• Light Microscopy
• Dark-field microscopes
• Best for observing pale objects
• Only light rays scattered by specimen enter objective lens
• Specimen appears light against dark background
• Increases contrast and enables observation of more
details

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Figure 4.6 The light path in a dark-field microscope.
Objective
Light refracted
by specimen
Light unrefracted
by specimen
Specimen
Dark-field stop
Condenser
Dark-field stop

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Microscopy
• Light Microscopy
• Phase microscopes
• Used to examine living organisms or specimens that
would be damaged/altered by attaching them to slides or
staining
• Light rays in phase produce brighter image, whereas light
rays out of phase produce darker image
• Contrast is created because light waves are out of phase
• Two types
• Phase-contrast microscope
• Differential interference contrast microscope

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Rays in phase Rays out of phase
Phase plate
Bacterium Ray deviated by
specimen is 1/4
wavelength out
of phase.
Deviated ray
is now 1/2
wavelength
out of phase.
Figure 4.7 Principles of phase microscopy.

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Figure 4.8 Four kinds of light microscopy.
Nucleus
Bacterium
Bright field Dark field
Phase contrast Nomarski

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Microscopy
• Light Microscopy
• Fluorescent microscopes
• Direct UV light source at specimen
• Specimen radiates energy back as a longer, visible
wavelength
• UV light increases resolution and contrast
• Some cells are naturally fluorescent; others must be
stained
• Used in immunofluorescence to identify pathogens and to
make visible a variety of proteins

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Figure 4.9 Fluorescence microscopy.

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Antibodies
Bacterium
Cell-surface
antigens
Bacterial cell with
bound antibodies
carrying dye
Fluorescent dye
Antibodies
carrying dye
Figure 4.10 Immunofluorescence.

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Microscopy
• Light Microscopy
• Confocal microscopes
• Use UV lasers to illuminate fluorescent chemicals in a
single plane
• Resolution increased because emitted light passes
through pinhole aperture
• Each image is "optical slice" through specimen
• Computer constructs 3-D image from digitized images

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Light Microscopy

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Microscopy
• Electron Microscopy
• Light microscopes cannot resolve structures closer than
200 nm
• Electron microscopes have greater resolving power and
magnification
• Magnifies objects 10,000x to 100,000x
• Detailed views of bacteria, viruses, internal cellular
structures, molecules, and large atoms
• Two types
• Transmission electron microscopes
• Scanning electron microscopes

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Figure 4.11 A transmission electron microscope (TEM).
Lamp
Light microscope
(upside down)
Column of transmission
electron microscope
Condenser
lens
Specimen
Objective lens
Eyepiece
Final image
seen by eye
Electron gun
Specimen
Objective lens
(magnet)
Projector lens
(magnet)
Final image on
fluorescent screen
Condenser lens
(magnet)

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Figure 4.12 Scanning electron microscope (SEM).
Electron gun
Magnetic
lenses
Primary
electrons
Secondary
electrons
Specimen
Specimen
holder
Vacuum
system
Beam
deflector coil
Scanning
circuit
Photo-
multiplier
Detector
Monitor

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Figure 4.13 SEM images.

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Electron Microscopy

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Microscopy
• Probe Microscopy
• Magnifies more than 100 million times
• Two types
• Scanning tunneling microscopes
• Atomic force microscopes

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Microscopy
• Probe Microscopy
• Scanning tunneling microscopes
• Passes metallic probe above specimen surface
• Measures the electron flow (tunneling current) to and from
the probe and the specimen's surface
• Atomic force microscopes
• Passes probe lightly on the specimen surface
• Deflection of laser beam translated into atomic
topography

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Figure 4.14 Probe microscopy.
EnzymeDNA

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Microscopy
• Tell Me Why
• Why is magnification high but color absent in an
unretouched electron micrograph?

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Staining
• Most microorganisms are difficult to view by bright-
field microscopy
• Coloring specimen with stain increases contrast
and resolution
• Specimens must be prepared for staining

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Figure 4.15 Preparing a specimen for staining.

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Staining
• Principles of Staining
• Dyes used as stains are usually salts
• Chromophore is the colored portion of the dye
• Acidic dyes stain alkaline structures
• Basic dyes stain acidic structures
• More common because most cells are negatively charged

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Staining
• Simple stains—composed of single dye
• Differential stains—use more than one dye
• Gram stain
• Acid-fast stain
• Endospore stain
• Histological stains
• Special stains—reveal specific structures
• Negative (capsule) stain
• Flagellar stain

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Figure 4.16 Simple stains.

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Figure 4.17 The Gram staining procedure.

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Figure 4.18 Ziehl-Neelsen acid-fast stain.

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Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis.

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Staining
• Differential Stains
• Histological stains
• Two common stains used for histological specimens
• Gomori methenamine silver (GMS) stain
• Hematoxylin and eosin (HE) stain

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Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae.
Bacterium
Capsule
Background
stain

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Flagella
Figure 4.21 Flagellar stain of Proteus vulgaris.

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Staining
• Staining for Electron Microscopy
• Chemicals containing heavy metals are used for
transmission electron microscopy
• Stains may bind molecules in specimens or the
background

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Staining

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Microscopy
• Tell Me Why
• Why is a Gram-negative bacterium colorless but a
Gram-positive bacterium purple after it is rinsed with
decolorizer?

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Classification and Identification of
Microorganisms
• Taxonomy consists of classification,
nomenclature, and identification
• Organize large amounts of information about
organisms
• Make predictions based on knowledge of similar
organisms

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Classification and Identification of
Microorganisms
• Linnaeus and Taxonomic Categories
• Current taxonomy system began with Carolus Linnaeus
• His system classified organisms based on characteristics
in common
• Grouped organisms that can successfully interbreed into
categories called species
• Used binomial nomenclature

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Figure 4.22 Levels in a Linnaean taxonomic scheme.

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Classification and Identification of
Microorganisms
• Linnaeus and Taxonomic Categories
• Linnaeus proposed only two kingdoms
• Later taxonomic approach based on five kingdoms
• Animalia, Plantae, Fungi, Protista, and Prokaryotae
• Linnaeus's goal was to classify organisms in order to catalog
them
• Modern goal is to understand relationships among organisms
• Goal of modern taxonomy is to reflect phylogenetic hierarchy
• Greater emphasis on comparisons of organisms' genetic
material led to proposal to add domain

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Classification and Identification of
Microorganisms
• Domains
• Carl Woese compared nucleotide sequences of rRNA
subunits
• Proposal of three domains as determined by ribosomal
nucleotide sequences
• Eukarya, Bacteria, and Archaea
• Cells in the three domains also differ with respect to
many other characteristics

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Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Physical characteristics
• Biochemical tests
• Serological tests
• Phage typing
• Analysis of nucleic acids

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Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Physical characteristics
• Can often be used to identify microorganisms
• Protozoa, fungi, algae, and parasitic worms can often
be identified based only on their morphology
• Some bacterial colonies have distinct appearance
used for identification

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Figure 4.23 Two biochemical tests for identifying bacteria.
No
hydrogen
sulfide
Hydrogen
sulfide
producedAcid with gas Acid with no gas Inert
Gas bubble Inverted tubes to trap gas

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Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system.
Wells

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Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Serological tests
• Serology—study of serum (liquid portion of blood after
clotting factors removed)
• Many microorganisms are antigenic
• Trigger immune response that produces antibodies
• Serum is an important source of antibodies
• Antibodies can be isolated and bind to the antigens that
triggered their production

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Figure 4.25 An agglutination test, one type of serological test.

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Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Phage typing
• Bacteriophage (phage)—virus that infects bacteria
• Phages are specific for the host they infect
• Phage typing is based on this specificity

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Bacterial lawn
Plaques
Figure 4.26 Phage typing.

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Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Analysis of nucleic acids
• Nucleic acid sequence can be used to classify and
identify microbes
• Prokaryotic taxonomy now includes the G + C content of
an organism's DNA

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Classification and Identification of
Microorganisms
• Taxonomic Keys
• Dichotomous keys
• Series of paired statements where only one of two
"either/or" choices applies to any particular organism
• Key directs user to another pair of statements or provides
name of organism

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Figure 4.27 Use of a dichotomous taxonomic key.

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Dichotomous Keys: Overview

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Classification and Identification of
Microorganisms
• Tell Me Why
• Why didn't Linnaeus create taxonomic groups for
viruses?